1. Field of the Invention
The present invention refers to a method for fluorescence spectroscopic determination of a biological substance provided with a marker consisting of a lanthanide chelate complex formed by a lanthanide coupled to the substance via a chelate forming compound, such as an EDTA-analogue.
2. Prior art
In quantitative immunological determinations an antibody or an antigen or its conjugate is usually provided with an easily detectable marker which at present usually consists of a radioactive isotope. The disadvantages of using radioactive isotopes are their limited storage time and the fact that one wishes to decrease the use of radioactive substances within medicine for health and environment reasons. An alternative to the radioimmunological methods are the fluoroimmunological methods where the marker is a fluorescent substance.
A special problem in fluorescence determinations within the immunological field is that serum has a relatively strong fluorescence which gives rise to high background levels for most fluorescence markers. This fluorescence of serum derives mainly from different proteins which however have relatively short excitation and emission wavelengths (excitation maximum at 280 nm, emission maximum at 340 nm) and thus it does not constitute a significant source of noise. Serum does however also give rise to other types of fluorescence, presumably deriving from other compounds than protein, this fluorescence appearing at longer wavelengths (excitation at 340 nm, emission at 460 to 470 nm) and thus gives rise to noise phenomena which are more difficult to deal with. In addition to the inherent fluorescence of serum the background is also affected by the scattering of the sample. The scattering gives rise to an interference, especially if markers with a small Stoke's shift (less than 50 nm) are used. Because of the high background fluorescence and the scattering of the fluorescence the sensitivity of markers is decreased by 50 to 100 times if used in serum as compared to their use in pure buffer.
Another problem arises in the fluorescence determination where the antibodies and the biologically active compound which is subject to the analysis are attached to a surface such as the wall of a test tube in addition to their internal coupling. Thus, the attached compounds have a strong inherent fluorescence and the surface made of glass or plastic is often also fluorescent and additionally has disturbing light scattering properties. The fluorescence measuring from a surface is thus more difficult from a measuring point of view than the measurement of a solution.
The requirements of a marker which is used in an immunological system are that it has to have as high a fluorescence as possible, a relatively long emission wavelength (more than 500 nm), a high Stoke's shift and the property of being coupled (covalently to the antibody or the antigen) without a negative effect on the conjugation properties.
A fluorescent marker meeting these requirements is described in the Swedish patent application No. 7902078-8 and is formed by a lanthanide chelate made up from a lanthanide and an aromatic .beta.-diketone, which is coupled to the compound via an EDTA-analogue so as to obtain a fluorescent lanthanide complex. This marker furthermore has the essential property of a long fluorescent time, 50 to 1000 .mu.sec which makes it possible to use an instrumentation technique described for instance in U.S. Pat. No. 4,058,732 in accordance with which the sample is excited by a laser pulse of a short duration, the fluorescence being detected only when the fluorescence from the noise sources has declined.
The disadvantage of using the above described marker is that the .beta.-diketones disturb the immunoreaction and when they are supplied after the termination of the immumoreaction a very long time is required for the formation of the ternary chelate and furthermore, the protein used is often coupled to a surface, for instance, the wall of a test tube which makes measurements of a sufficient accuracy difficult to carry out.